C1q is a component of the C1 complex of the classical complement pathway (R. B. Sim and K.B.M. Reid, Immunology Today 1991; 12:307-311). The biological functions of C1q are diverse, including initiation of the complement cascade for opsonization and cytolysis, and mediation of several different functions depending on the cell types expressing the C1q receptor. C1q enhances FcR and CRl--mediated phagocytsis in monocytes/macrophages (D. A. Bobak et al., Eur. J. Immunol. 1988; 18:2001-2007; D. A. Bobak et al., J. Immunol. 1987; 138:1150-1156), stimulates immunoglobulin production by B cells (K. R. Young et al., J. Immunol. 1991; 146:3356-3364), activates platelets to express .alpha.IIb/.beta..sub.3 integrins, P-selectin, and procoagulant activity (E.I.B. Peerschke et al., J. Exp. Med. 1993; 178:579-587; E.I.B. Peerschke et al., J. Immunol. 1994; 152:5896-5901), activates tumor cytotoxicity of macrophages (R. W. Leu et al., J. Immunol. 1990; 144:2281-2286), and exerts anti-proliferative effects on T cell growth (A. Chen et al., J. Immunol. 1994; 153:1430-1440).
A 33 kilodalton (kD) receptor, designated gC1q-R, which binds to the globular head of C1q molecules has been recently identified, cloned and sequenced (B. Ghebrehiwet et al., J. Exp. Med. 1994; 179:1809-1821; E.I.B. Peerschke et al., J. Immunol. 1994; 152:5896-5901; A. Chen et al., J. Immunol. 1994; 153:1430-1440). Another 60 kD receptor, designated cC1q-R, binds to the amino-terminal collagen-like region of C1q (B. Ghebrehiwet, Behring Inst. Mitt. 1989; 84:204-215; A. Chen et al., J. Immunol. 1994; 153:1430-1440). Based on the detection of gC1q-R mRNA by polymerase chain reaction (PCR) amplification and gC1q-R protein expression by immunochemical methods, this receptor was found to exist on a large number of different cell types, e.g. B cells, T cells, monocytes/macrophages, neutrophils, eosinophils, fibroblasts, platelets, endothelial cells, liver cells, neural cells and smooth muscle cells. It was not known, however, that gC1q-R binds to HIV-1gp120 and neutralizes the infectivity of HIV-1.
It is well established that the CD4 antigen, which is expressed mainly on the surface of the helper/inducer T cells, is the primary receptor for HIV-1 gp120 (P. J. Maddon et al., Cell 1986; 47:333-385; J. S. McDougal et al., Science 1986; 231:382-385). In addition to the CD4.sup.+ T cells, HIV-1 can bind to and infect a number of other cell types, such as monocytes/macrophages, B cells, colon eptihelial cells and neuroglial cells, which express either undetectable or at most low levels of cell-surface CD4. Several alternative receptors have been suggested to be associates with HIV-1 infection of target cells, e.g. galactosylceramide (Gal-Cer) on the surface of human colon epithelial cells, Schwann cells, and oligodendrocytes (N. Yahl et al., Virology 1994; 204:550-557; J. M. Harouse et al., Science 1991; 253:320-323), human immunoglobulin V.sub.H 3 gene products of IgM isotype (mol. wt. 950 kD) on B cells (L. Berberian et al., Science 1993; 261:1558-1591) CD26 (mol. wt. 110 kD) on activated T and B cells (C. Callebaut et al., Science 1993; 262:2045-2050), and a membrane-associates C-type lectin (mol. wt. 46 kD) mainly on macrophages (B. M. Curtis et al., Proc. Natl. Acad. Sci. USA 1992; 89:8356-8360).
This invention was made in the course of research to find cellular binding proteins of receptors for HIV-1 gp120 on non-CD4 expressing cells. To this end, lysates of CEM-SS cells (which are T cells expressing a high level of cell-surface CD4) obtained from P. J. Nara (AIDS Res. Hum. Retroviruses 1987; 3:283-302) and DAKIKI cells (which are CD4-negative B cells) obtained from American Type Culture Collection (Rockville, Md.) were run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the separated proteins were transblotted onto nitrocellulose membranes for Western immunoblot assays. It was found that reombinant or HIV-1 --infected cell derived gp120 reacted with a protein band of 32-33 kD in both CEM-SS and DAKIKI cell lysates, which was distinct from the 55 kD protein band reactive with anti-human CD4 monoclonal antibodies.
In order to purify this novel gp120-binding protein for identification by N-terminal amino acid sequencing, a large quantity of DAKIKI cell lysate was prepared. The gp120--binding protein was partially purified by preparative electrophoresis using Prep Cell Model 491 (Bio-Rad Laboratories, Inc., Hercules, Calif.). The sample was then run on 2-dimensional gel electrophoresis and blotted onto polyvinylidene difluoride (PVDF) membrane for Western immunoblotting to identify the gp120--reacting protein spot and for N-terminal amino acid sequencing. It was determined that the sequence of the first fifteen amino acids at the N-terminus of the gp120--reacting protein was identical to that of the protein previously identified as p32 (mol. wt. 32 kD), which was co-purified with a human pre-mRNA splicing factor by A. R. Krainer et at. (Cell 1991; 66:383-394) and B. Honore et al. (Gene 1993; 134:282-287). Further experiments revealed that DAKIKI cells can be stained by rabbit anti-p32 immunoglobulins which were generated by using purified E. coli expressed p32 as the immunogen, showing that p32 exists on the cell surface of DAKIKI cells. DAKIKI cells were also shown to bind recombinant HIV-1 gp120, even though the cells do not express detectable CD4 on the cell surface by immunofluorescence methods. Taken together, these findings show that p32 is an alternative cell-surface binding protein for HIV-1 gp120. Subsequently, it was determined that p32 has the same sequence as gC1q-R (SEQ ID NO.: 1) (B. B. Ghebrehiwet et al., J. Exp. Med. 1994; 179:1809-1821).
The precursor or pre-propotein of gC1q-R has 282 amino acids (a.a.), and the functional mature protein has 209 a.a.. This mature protein contains the peptidic portion between amino acid residue nos.: 74 (leucine) and 282 (glutamine), of SEQ ID NO.: 1. Mature gC1q-R protein is highly charged and acidic. It has three potential N-glycosylation sites at amino acid positions: 114, 136 and 223. As a result, the apparent molecular weight of gC1q-R in SDS-PAGE is 32 kD, instead of 24.3 kD as calculated from the amino acid composition. Since the mature protein has only one cysteine residue at position 186, there is no intrachain disulfide linkage within the protein. gC1q-R is found in a wide variety of cell types, such as B cells, T cells, monocytes/macrophages, esosinophils, neutrophils, platelets, endothelial cells, fibroblasts, and liver cells.
Inasmuch as gC1q-R is associated with multiple immunological and physiological functions, the interaction between gC1q-R and HIV-1 gp120 may account for some of the immunological and physiological dysfunctions manifested by HIV-1 -infected individuals. It may also result in the known ability of HIV-1 to infect a side variety of non-CD4 expressing human cells in different tissues and organs. Intervention in the interaction between gC1q-R and HIV-1 gp120 represents a new strategy in combating HIV-1 disease. Several inventions based on this intervention strategy are discussed below.